The Effects Of VX-680 Combined With Cisplain On The Proliferation,Apoptosis And Migration In Esophageal Cells

Objective:Investigate the effect of Aurora protein inhibitor kinase VX-680 combined with cisplain on the proliferation,apoptosis,migration and angiogenesis in human esophageal cell line EC9706 and its molecular mechanism.Methods:1.The effects of VX-680 and cisplain alone and combination on proliferation,apoptosis,adhesion,migration and tube formation of human esophageal cell line EC9706.respectively,different concentrations of VX-680,CDDP,VX-680+CDDP were treated on the cells for 48 h.and the morphological changes of the cells were observed by inverted microscope.The cell viability was detected by MTT assay.And Combination index(CI)was calculated by Chou-Talalay method.The apoptosis was detected by intercelluar and extracellular adhesion experiments.The cell migration was detected by scratch test.The effect of esophageal cells on angiogensis was detected by tube formation assay.2.The effect of VX-680 and cisplain alone and combination on apoptosis,adhesion,migration and tubule formation related protein of human esophageal cell line EC9706.the cells were treated with different concentrations of VX-680,CDDP,VX-680+CDDP for48 h.the apoptosis protein PARP,caspase-3,adhesion moleculor,E-cadherin,and the molecular metallproteinase MMP-2 which affect migration,and the expression of VEGF,were detected by Western blot,3.Western blot results showed that the expression levels of p-ERK and p-AKT in ERK and AKT signally pathways were down-regulated in the VX-680+CDDP treated group compared with the control and monotherapy groups.While the total protein ERK and AKT did not significantly changed.Results:1.In the VX-680 +CDDP treatment group,a large number of esophageal cancer cells EC9706 floating,a small amount of adherent,the inhibition rate of cell proliferation was(68% ±0.14%),while in the single drug group the inhibition rate was(43% ±0.02%),(15%±0.04%),and the two drugs were synergistic.DAPI staining showed that the proportion of nuclear condensation and nuclear fragmentation was significantly higher than that of single drug group(P<0.05).The intercellular adhesion experiment showed that the VX-680 +CDDP group was larger and more compact than the single-drug group(P<0.01).The degree of intercellular dispersion is weaker;The cell-extracellular matrix adhesion experiment showed that compared with the monotherapy group,the cell adhesion rate in the VX-680 +CDDP treatment group was significantly reduced in the three different extracellular matrix(P<0.05).Scratch test results showed that the cell healing ability of VX-680 +CDDP group was weaker than that of single drug group(P<0.001).Tubule formation in HUVEC cells treated with VX-680 +CDDP was significantly lower than that treated with monotherapy(P<0.001).2.Esophageal cancer cells EC9706 VX-680,CDDP,VX-680 + CDDP treatment after48 h,Caspase-3 proenzyme protein involved in apoptosis and the expression of PARP original belt level significantly decreased in the blank control group and drug group,procaspase 3,cleaved-PARP raised obviously,adhesion molecules E-cadherin expression level rise,in the control group and single drug group transfer related molecular MMP-2expression is abate,VEGF expression level lowered in the control group and single drug group.3.Western blot results showed that in the VX-680 +CDDP treatment group,the expression levels of p-ERK and p-AKT in the ERK and AKT signaling pathways were down-regulated compared with the control group and the single-drug group,while the total proteins ERK and AKT were not significantly changed.Conclusion1.The combination of VX-680 and cisplain inhibited the proliferation of esophageal cancer cells,promoted apoptosis,increased cell-cell homogenous adhesion,reduced cell-extracellular matrix adhesion.Inhibited migration and weaken the angiogenic capacity of HUVEC.2.The combination of VX-680 and cisplain could promot the apoptosis of esophageal cancer EC9706 by regulating the activation of caspase-3 and PARP,the combination of VX-680 and cisplain increased cell-cell adhesion but reduce cell-extracelluar adhesion by enhancing E-cadherin the migration ability of EC9706 cells and the angiogenic ability of HUVEC cells were reduced by down-regulating MMP-2expression and VEGF in esophageal cells.3.The combination of VX-680 and cisplain may affect the malignant phenotype of EC9706 cells through AKT and ERK signaling pathway.